The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Potent cryoprotective activity of cold and CO2-regulated cherimoya (Annona cherimola) endochitinase.

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO(2)-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M(r) of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 degrees C, and it retained about 30% of its maximum activity at 5 degrees C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 degrees C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k(cat) (6.93 s(-1)) value for the fluorogenic substrate 4-MU-(GlcNAc)(3), reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k(cat) at 37 and 5 degrees C, and a low E(a) (11.32 kJ mol(-1)). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze-thaw inactivation of lactate dehydrogenase. The PD(50) was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO(2)-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO(2) concentrations.

High CO2 atmosphere modulating the phenolic response associated with cell adhesion and hardening of Annona cherimola fruit stored at chilling temperature.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) activity, tanning ability, and polyphenols levels were measured in cherimoya (Annona cherimola Mill.) fruit treated with 20% CO(2) + 20% O(2) + 60% N(2) for 1, 3, or 6 days during chilling temperature (6 degrees C) storage. The residual effect of CO(2) after transfer to air was also studied. These observations were correlated with texture and cellular characteristics, visualized by cryo-SEM. Tanning ability and the early increase in tannin polyphenols induced by chilling temperature were reduced by CO(2) treatment. Conversely, high CO(2) atmosphere enhanced the nontannin polyphenol fraction as compared with fruit stored in air. Lignin accumulation and PAL activation observed in untreated fruit after prolonged storage at chilling temperature were prevented by high CO(2). Moreover, the restraining effect on lignification was less effective when the CO(2) treatment was prolonged for 6 days. In addition, fruits held at these conditions had greater firmness and the histological characterization of the separation between cells was similar to that in untreated fruits. We conclude that CO(2) treatment modulates the phenolic response that seems to regulate the strength of cell adhesion and so to prevent hardening caused by chilling temperature storage.